There is increasing interest in the use of protein hydrolysates for both medical and non-medical applications. In both applications an easily assimilable diet featuring facilitated gastrointestinal uptake of proteins is a factor of prime importance. Protein hydrolysates for medical applications also require strongly reduced allergenic properties. For products intended for non-medical applications, good taste characteristics and good solubilities under acid conditions are important characteristics. Unfortunately the hydrolysis process required to realise these benefits comes with a number of disadvantages. These include bitter off-tastes, residual immunogenic materials, low yields of nutritionally indispensible amino acids, high osmotic values caused by the release of free amino acids and, finally, limited acid stabilities.
In prior publications several enzyme mixtures aimed at optimising hydrolysate characteristics and lowering production costs have been described. All of these publications refer to the use of single or mixed endoproteases. Examples include EP 321 603, which refers to the use of animal-derived endoproteases like trypsin, chymotrypsin and pancreatin, and EP 325 986 and WO 96/13174, which favor the use of endoproteases obtained from Bacillus or Aspergillus species. Unfortunately these enzyme combinations always yield peptide mixtures which are bitter and exhibit a broad molecular weight distribution. Large molecular weight peptides are undesirable because they are responsible for the allergenic response and their uptake requires additional enzymatic processing steps in the intestine. Reducing the bitter off-taste in the hydrolysates makes the use of exoproteases such as aminopeptidases or carboxypeptidases indispensible. Disadvantages of this debittering process are the release of substantial quantities of free amino acids and thus brothy off flavors and losses of nutritionally important amino acids.
In conclusion, industrial production of protein hydrolysates continues to rely on enzyme mixtures which are far from optimal so that expensive purification steps are needed to produce peptide mixtures having sub-optimal size distributions.
Upon normal dietary intake the proteins present in food are gradually hydrolysed to smaller fragments which are finally transported across the wall of the small intestine. During passage through the gastrointestinal tract a number of different proteases that originate in the stomach, pancreas and small intestine are active. Endoproteases such as pepsin, trypsin and chymotrypsin cleave the large molecular weight proteins into smaller oligopeptides. These oligopeptides are then further hydrolysed by a number of other enzymes such as di- and tripeptidyl peptidases as well as amino- and carboxypeptidases. The final steps of hydrolysis take place in the small intestine and result in a mixture of free amino acids and di- and tripeptides (Grimble, G. K. 1994. Annu. Rev. Nutr. 14; 419-447).
Despite the large collection of proteases that is active in the gastrointestinal tract, it is likely that peptides that resist further proteolytic hydrolysis in the small intestine form a major fraction of the surviving population of di- and tripeptides. It has, for example, been reported that di- and tripeptides carrying carboxyterminal proline residues exhibit stabilities in the body which are up to 3 orders of magnitude higher than other peptides (Ashmarin, I. P. et al.; Biochemistry (Moscow), Vol 63, No 2, 1998, pp 119-124). Carrier systems specific for the transport of either the free amino acids or the di- and tripeptides are responsible for the efficient transport across the intestine wall. A peptide sequence-independent mechanism capable of transporting quantitatively significant amounts of intact di- and tripeptides has been identified (Doering, F. et al; 1998; J. Biol. Chem. 273, 23211-23218). After entering the blood circulation, the peptides may potentially act as physiological modulators of metabolism. The physiological effects of peptides with opioid, ACE-inhibitory, antithrombosis, antiulcer, antiarthritic and anorectic activities have been described (Pihlanto-Leppala, A; Trends in Food Science & Technology 11 (2001) 347-356; Ashmarin, I. P. et al.; Biochemistry (Moscow), Vol 63, No 2, 1998, pp 119-124).
The recent commercialisation of various protein hydrolysates claiming antihypertensive effects emphasize the increased scope of use of protein hydrolysates containing “bioactive” peptides in medical and non-medical applications. These bioactive peptides and protein hydrolysates containing such bioactive peptides have been described in a number of patent applications. For example, WO 97/00078 describes hydrolysates obtained by incubation with probiotic bacteria or enzymes obtained from such bacteria. WO 99/16461 describes the inhibition of angiotensin-converting enzyme by specific tripeptides obtained by fermentation of Lactobacillus. WO 01/32905 describes the preparation of a product containing antihypertensive peptides by fermenting casein with lactic acid bacteria. Several other applications (see for example WO 01/68114) describe the use of highly purified or chemically synthesized peptides for reducing blood pressure or treating diabetes, renal impairment or obesity.